The long-term goal of this U01 proposal is to develop Thermoresponsive (TR)-NanoVelcro circulating tumor cell (CTC) purification system that can be digitally programmed to achieve optimal performance for recovering viable CTCs in prostate cancer (PC) patients' blood, allowing seamless coupling with various downstream functional and molecular assays. Dr. Tseng (PI/UCLA) and Dr. Posadas (co-PI/Cedars Sinai Medical Center) will bring together an interdisciplinary research team to implement the proposed research activities. CTCs are regarded as a liquid biopsy of tumors, allowing non-invasive, repetitive, and systemic sampling of the disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic PC, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal PC. In order to obtain CTC-derived molecular signatures and functional readouts, it is important to develop improved methodologies that can not only detect/enumerate CTCs with high sensitivity, but also recover CTCs with minimum contamination by white blood cells and negligible disruption to CTCs' viability. Our working hypothesis, based on preliminary data gathered of temperature-dependent purification of viable CTCs using polymer brush-grafted nanosubstrates, is that the performance of the proposed TR- NanoVelcro CTC purification system can be i) optimized by rationally modulating surface chemistry, cocktail capture agents, flow rates, and heating/cooling cycles, and ii) validated using both artificial and prostate cancer patient blood samples. PC CTCs purified by TR-NanoVelcro CTC purification system will be of sufficient viability and purity, paving the way for i) short-term in vitro culture, ii) long-term i vitro maintenance, and iii) in vivo tumorigenic models as patient-derived xenografts. The ex vivo expanded CTCs will provide sufficient high-quality gDNA and mRNA that can be characterized by next-generation sequencing (NGS) for de novo identification of key molecular events in PC. Using bioinformatic approaches, we will assemble PC-specific genomic/transcriptomic panels for cross-validation by NGS using CTCs freshly isolated from multiple PC patients. We envision that TR-NanoVelcro system will enable instant purification of viable CTCs from PC patients, paving the way for performing a variety of downstream molecular and functional assays that can significantly contribute to understanding PC progression, implementation of personalized treatment, and development of new therapeutics.